The present invention relates to analytical systems and, more particularly, to chemical analysis instruments in which sample components are separated by differential migration rates through a narrow-bore capillary. A major objective of the present invention is to provide for both high-resolution component separation and high-sensitivity detection of separated components of proteins and comparable complex species.
Advances in biotechnology have relied in large part on techniques of chemical analysis. Biotechnology has provided techniques for manufacturing life-supporting medicines and other products which would otherwise be in short supply if natural sources had to be relied upon. In addition, entirely new medical products are in development which may arrest and cure heretofore untreatable diseases. Biotechnology promises new products for agriculture which will feed the world's expanding populations and which will enhance the ability of famine-prone countries to sustain themselves.
Chemical analysis of the proteins found in biological samples generally involves the separation of the samples into components for identification and quantification. Capillary zone electrophoresis (CZE) is one of a class of methods in which the different components are moved within a narrow-bore capillary at respective and different rates so that the components are divided into distinct zones. The distinct zones can be investigated within the capillary or outside the capillary by allowing the components to emerge from the capillary for sequential detection.
In CZE, a sample is introduced at an input end of a longitudinally extending capillary and moved toward an output end under the influence of an electric field. This influence combines two electro-kinetic effects: electro-osmotic flow and electrophoretic migration.
Electro-osmotic flow results from charge accumulation at the capillary surface due to preferential adsorption of anions from the electrolyte solution that fills the capillary bore. The negative charge of the anions attracts a thin layer of mobile positively charged electrolyte ions that accumulate adjacent to the inner surface. The thin layer of mobile positively charged electrolyte ions is pulled toward the negative electrode, dragging the bulk of the sample along with it. Thus, the electro-osmotic flow results in a mean flow of sample components from the positive electrode toward the negative electrode.
Superimposed on this electro-osmotic flow is electrophoretic migration, the well-known motion of charged particles in an electric field. The electrolyte solution acts as the medium that permits the electric field to extend through the capillary between the electrodes. Positively charged molecules are attracted toward the negative electrode so they flow faster than the mean flow determined by the electro-osmotic flow. Negatively charged molecules are attracted toward the positive electrode. For the negatively charged particles, the electrophoretic flow components opposes the, generally larger, electro-osmotic flow component. The result is that negatively charged particles flow toward the negative electrode, but more slowly than the mean flow.
As a result of the combined electrophoretic and electro-osmotic flow, each sample component moves through the capillary separation column at a rate dependent on its species-specific charge. Due to the differential flow rates, the components separate after a sufficiently long migration through the separation capillary. An appropriately selected and arranged detector can detect these zones seriatim as they pass. Components can be identified by the time of detection and can be quantified by the corresponding detection peak height and/or area. In some cases, the bands can be collected in separate containers for a distinct identification and/or quantification process.
There are several types of detectors used to detect proteins in capillary separation systems. Ultraviolet absorbance (UV) detectors are among the most common. In addition, chemiluminescence, refractive index, and conductivity detectors have been used. All these methods lack the sensitivity required to detect many peaks obtained in CZE protein analysis.
High sensitivity is required because the quantity of the total sample is limited, and the detector must be capable of detecting components that make up only a fraction of the total sample. Limitations on sample quantity stem from the requirement that the sample be dissolved in electrolyte and that the concentration of the sample be low enough to avoid perturbation of the electrical field which would lead to distortion of the separated component zones. The sample quantity is further limited by the capillary bore diameter and by the necessity of confining the sample initially to a relatively short longitudinal extent. The initial sample extent governs the minimum zone breadth and thus the ability of the detector to resolve similarly charged sample components.
The detector must be able to detect small quantities of the component in each sample zone. A UV detection system faced with low concentrations and a short illumination path across a capillary typically yields a poor signal-to-noise ratio. Other detection methods are similarly limited. Thus, while CZE is effective in separating protein components, it has been difficult to find a sufficiently sensitive detector for identifying and quantifying the separated components.
Fluorescence detection has been applied in conjunction with liquid chromatography (LC), a class of alternative component separation techniques. In liquid chromatography, a liquid mobile phase ushers components through a capillary at different rates related to the components' partitioning between the mobile phase and a stationary phase. Zones thus form as a function of partitioning ratios. The zones can be illuminated and the resulting fluorescence detected. Few proteins can be detected with sufficient sensitivity using their intrinsic fluorescence. However, labeling reagents can be used to enhance protein fluorescence. A major advantage of using fluorescence detection is that the increased sensitivity required by small sample quantities can be achieved by using very intense illumination. Thus, fluorescence detection used with labeling reagents promises to enhance the ability to identify and quantify sample components.
Unfortunately, liquid chromatography is not well suited for high resolution separation of proteins. While partitioning ratios differ among components, the molecules of any one component at any time are divided between the mobile phase and the stationary phase, and thus move at different rates relative to each other. Despite averaging effects over the length of the capillary, sufficient zone broadening is induced by the partitioning to prevent high resolution separation of protein components. Since its only source of zone broadening is longitudinal diffusion, CZE represents an approximately ten-fold improvement in zone-breadth-limited resolution over liquid chromatography.
Fluorescence detection of proteins is not generally used in conjunction with CZE for a number of reasons. As indicated above, few proteins can be detected with sufficient sensitivity using their intrinsic fluorescence. Pre-separation fluorescence labeling is incompatible with CZE since it causes same-species molecules to have different charges. Thus, one component separates into multiple peaks, rendering detections virtually uninterpretable. Furthermore, sensitivity problems are compounded because each peak represents only a fraction of a sample component.
Post-separation labeling involves the introduction of a fluorogenic labeling reagent after separation and before detection. Post-separation mixing is addressed by Van Vliet et al., "Post-Column Reaction Detection for Open-Tubular Liquid Chromatography Using Laser-Induced Fluorescence," Journal of Liquid Chromatography, Vol. 363, pp. 187-198, 1986. This article discloses the use of a Y-connector for introducing reagent into the effluent of a separation capillary. One problem with the Y-connector is the inevitable turbulence that occurs as the streams merge at an oblique angle. The turbulence stirs the sample stream, severly broadening the component zones. This broadening can be tolerable in a low-resolution system, but not in a high-resolution CZE system.
Post-separation mixing is also addressed by Weber et al. in "Peroxyoxalate Chemiluminescence Detection with Capillary Liquid Chromatography" in Analytical Chemistry, Vol. 59, pp. 1452-1457, 1987. Weber et al. disclose the use of a Teflon tube to convey the separated sample components emerging from a liquid chromatography capillary, packed with silica particles to the interior of a mixing capillary. An annular gap between the Teflon tube and the mixing capillary is used to introduce the chemiluminescence reagent coaxially of the sample emerging from the narrower (0.2 mm) Teflon tube and into the (0.63 mm) mixing capillary. Turbulence is minimized since the reagent flow is fast enough to define a sheathing flow confining the sample.
A major limitation of the approach of Weber et al. is that chemiluminescence cannot generally be employed in protein component detection. Many proteins cannot activate chemiluminescence reagents. Moreover, the approach does not provide the required sensitivity for those proteins that do activate the reagents.
Another problem with the approach of Weber et al. is that the sheathing flow causes mixing to occur slowly. Sufficient mixing of the chemiluminescence reagent with sample components thus requires a relatively long mixing interval and large mixing volume, resulting in substantial zone broadening. This zone broadening significantly impairs resolution. While this zone broadening may be tolerable in the relatively low resolution liquid chromatography system disclosed, it would negate the advantages of a high-resolution CZE system.
Another approach is to narrow the effluent end of the separation capillary so that it can fit inside the input end of the mixing capillary. When a tapered separation capillary is fit inside a constant bore size mixing capillary, an annular gap is created between the capillaries. Fluorescent labeling reagent can then be introduced through the annular gap. However, this approach has limitations. For optimal fit, the mixing capillary has an inner bore diameter greater than the inner bore diameter of the separation capillary. When the sample exits from the smaller bore and enters the larger bore, the sample spreads, leading to broadened sample zone widths.
Other approaches have involved the introduction of fluorescent labeling reagents through apertures in capillary walls. In a CZE separation system, an aperture or other inhomogeneity in the capillaries defining the sample path can cause field perturbations, which can interfere with electro-osmotic and other electro-kinetic effects. At a minimum, these perturbations cause zone broadening, but can even partially or completely impair electro-kinetic movement of sample components.
In summary, CZE provides a separation technique which affords the resolution required for the analysis of complex proteins, but lacks a sufficiently sensitive compatible detection technique. Fluorescence detection provides a desirable level of sensitivity, but the required labeling has not been workable in the CZE context. What is needed is a system that combines the resolving power of CZE with the detection sensitivity available with fluorescence-labeled proteins.